Compositions useful in treatment of ornithine transcarbamylase (otc) deficiency

ABSTRACT

Viral vectors comprising engineered hOTC DNA and RNA sequences are provided which when delivered to a subject in need thereof are useful for treating hyperammonemia, ornithine transcarbamylase deficiency and symptoms associated therewith. Also provided are methods of using hOTC for treatment of liver fibrosis and/or cirrhosis in OTCD patients by administering hOTC.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/191,709, filed Nov. 15, 2018, which is a continuation of U.S. patentapplication Ser. No. 15/122,853, filed Aug. 31, 2016, now U.S. Pat. No.10,167,454, issued Jan. 1, 2019, which is a national stage applicationunder 35 U.S.C. 371 of PCT/US2015/019513, filed on Mar. 9, 2015, nowexpired, which claims the benefit of U.S. Patent Application No.61/950,157, filed Mar. 9, 2014, now expired. These applications areincorporated by reference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with support under grant Nos. P01-HD057247,P01-HL059407, and P30-DK047757 awarded by the National Institutes ofHealth. The US government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Ornithine transcarbamylase (OTC) deficiency accounts for nearly half ofall cases of inborn errors of urea synthesis, with a prevalenceestimated to be at least 1 in 15,000. Urea cycle defects put patients atrisk of life threatening elevation of ammonia that can lead toirreversible cognitive impairment, coma and death. Newborn males withcomplete deficiency develop hyperammonemic coma within the first 3 daysof life, which if untreated, is lethal.

Current therapies for OTC deficiency (OTCD) have numerous challenges.Patients can be managed with a low protein diet in combination with theuse of medications that activate alternate nitrogen clearance pathways,but this does not prevent hyperammonemic crises. Despite the use ofdialysis and alternate pathway therapy, there is almost a 50% mortalityrate in neonates. Liver transplantation can cure OTCD, but donor liveris limiting, the procedure carries significant morbidity andimmunosuppressive drugs are necessary for the duration of the subject'slife.

Gene therapy of a metabolic disease such as OTCD presents a morechallenging model for gene replacement therapy than other conditions.Because the gene acts in a cell-autonomous manner (i.e., it can onlyinfluence the cell in which it is expressed), therapeutic effects shouldbe directly correlated with the number of target cells that aretransduced, rather than with the net level of expression in liver suchas with a secreted protein where high expression per cell can overcomelow transduction. Furthermore, there has been at least one publishedreport that hOTCwt mRNA is unstable. [Wang, L., et al, MolecularGenetics and Metabolism, 105 (2012) 203-211].

There have been published reports of using viral vectors to try to treatOTC deficiency. For example, several groups have tried this in murinemodels of OTC deficiency, using recombinant adenoviruses carrying rat,mouse, or human OTC cDNA. Some measure of successful reconstitution ofliver OTC activity and correction of metabolic derangements have beenreported in animal models with viruses carrying rat or mouse OTC cDNA.Previous studies using adenoviral vectors have illustrated thedifficulties of expressing sufficient levels of active human OTC in OTCDmice.

Therefore, there is a need for other approaches to OTCD therapy.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a recombinant viral vector havingan expression cassette comprising an engineered nucleic acid sequenceencoding human ornithine transcarbamylase (hOTCase) and expressioncontrol sequences which direct expression of hOTC in a liver cell,wherein the hOTC nucleic acid sequence is less than 80% identical to thewild-type hOTC sequence over the full-length hOTC of the wild-typesequence (e.g., SEQ ID NO:1), or a fragment thereof which comprises themature hOTC but lacking at least the native leader sequence, or anotherintermediate which comprises at least the mature hOTC and expresses afunctional hOTCase. Suitably, the engineered sequence has beenpreferably codon optimized and further improved such that it enhances atleast one of transduction, transcription and/or translation of theenzyme.

The nucleic acid sequence may comprise the mature hOTC of SEQ ID NO: 5,or a nucleic acid sequence at least about 96 to about 99% identicalthereto or a nucleic acid sequence comprising at least the mature hOTCof SEQ ID NO: 9, or a nucleic acid sequence at least about 96 to about99% identical thereto, which expresses afunctional hOTCase. In oneembodiment, the hOTC is the full-length of SEQ ID NO: 5 or a nucleicacid sequence at least about 96 to about 99% identical thereto or anucleic acid sequence of the full-length of SEQ ID NO: 9, or a nucleicacid sequence at least about 96 to about 99% identical thereto. The hOTCsequence may be that of the corresponding nucleotides of SEQ ID NO: 3,4, 8 or 9. Encompassed within the scope of the invention are the strandscomplementary to those in the sequence listing. The viral vector may beselected from an adeno-associated virus (AAV) vector, an adenoviralvector, and a lentiviral vector.

In a further aspect, the invention provides a recombinantadeno-associated virus (rAAV) having an AAV capsid and packaged thereinan expression cassette comprising at least one AAV inverted terminalrepeat (ITR) sequence, an engineered nucleic acid sequence encodinghuman ornithine transcarbamylase (hOTCase) and expression controlsequences which direct expression of hOTC in a liver cell, saidexpression control sequences comprising a liver-specific promoter. Theengineered hOTC nucleic acid sequence is less than 80% identical to thewild-type hOTC sequence over the mature sequence or full-length hOTC ofthe wild-type sequence (e.g., SEQ ID NO:1) and expresses a functionalhOTCase. The synthetic hOTC nucleic acid sequence comprises at least themature hOTC of SEQ ID NO: 5 or a nucleic acid sequence at least about 96to about 99.9% identical thereto or a nucleic acid sequence comprisingat least the mature hOTC of SEQ ID NO: 9 or a nucleic acid sequence atleast about 96 to about 99.9% identical thereto.

In still a further aspect, the invention provides a viral vectorcomprising a hOTC gene encoding a chimeric ornithine transcarbamylasewhich comprises mature human ornithine transcarbamylase with aheterologous transit peptide, wherein the coding sequence from themature human ornithine transcarbamylase is selected from the nucleicacid sequence comprising at least the mature hOTC of SEQ ID NO: 3, 4, 5,8 or 9.Optionally, the full-length sequences of any of these sequences,which include the transit sequence, may be selected. Alternatively, achimeric OTC gene including a heterologous transit sequence, asdescribed herein, and these mature hOTC may be selected.

In another aspect, a pharmaceutical composition comprising a carrier andan effective amount of a vector as described herein is provided.

Yet another aspect is a viral vector as described herein used inpreparing a medicament for delivering ornithine transcarbamylase to asubject in need thereof and/or for treating ornithine transcarbamylasedeficiency. In one particularly desirable embodiment, the subject is ahuman subject. The subject may be homozygous or heterozygous forornithine transcarbamylase deficiency.

In still another aspect, use of a viral vector comprising a nucleic acidsequence encoding functional human ornithine transcarbamylase inpreventing and/or treating fibrosis or ornithine transcarbamylasedeficiency (OTCD)-related cirrhosis in a subject for OTCD is provided.In one embodiment, the subject is a human patient. In a furtherembodiment, the subject is heterozygous and may exhibit late onset ofsymptoms.

In yet a further aspect, use of a viral vector comprising a nucleic acidsequence encoding functional human ornithine transcarbamylase inpreventing and/or treating hepatocellular carcinoma in a subject havingOTCD is provided. In one embodiment, the subject is a human patient. Ina further embodiment, the subject is heterozygous for OTCD and mayexhibits late onset of symptoms.

Other aspects and advantages of the invention will be readily apparentfrom the following detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A provides a wild-type hOTC cDNA, which has 324 A, 223 C, 246 G,and 269 T [SEQ ID NO: 1].

FIG. 1B-1C provides the human ornithine transcarbamylase sequenceencoded by the sequence of FIG. 1A [SEQ ID NO:2].

FIG. 2 provides an engineered hOTC cDNA, with an altered GC ratio. Thebase count in the sequence is 283 A, 285 C, 284 G, and 216 T [SEQ ID NO:3].

FIG. 3 provides an engineered hOTC cDNA termed LW3 (SEQ ID NO:4). Thebase count in this sequence is 279 A, 303 C, 288 G, and 220 T. The startcodon for the hOTC open reading frame (ORF) is preceded by a Kozaksequence in this figure. The coding sequence for the leader begins atnucleotide 15 (first 96 nucleotides), followed by the coding sequencefor the 322 amino acid hOTCase. In this figure the stop codon isfollowed by a NotI restriction site (GCGGCCGC) which is a remnant of thevector.

FIG. 4 provides an engineered hOTC cDNA termed LW4 (SEQ ID NO: 5). Thebase count in this sequence is 278 A, 303 C, 289 G, and 220 T. Thecoding sequence for the leader begins at nucleotide 15 (first 96nucleotides), followed by the coding sequence for the 322 amino acidhOTCase. In this figure the stop codon is followed by a NotI restrictionsite (GCGGCCGC) which is a remnant of the vector.

FIGS. 5A-5C provides an alignment of the cDNA sequences of the wild-typehOTC, and five engineered sequences, hOTCco (SEQ ID NO: 3), LW3 (SEQ IDNO: 4), LW4 (SEQ ID NO: 5), LW5 (SEQ ID NO: 8) and LW6 (SEQ ID NO: 9).The aligned sequences contain a Kozak sequence (first 14 nucleotides ofLW3 and LW4) and a restriction enzyme site (following termination codonfor LW3 and LW4), which are not part of the open reading frame.

DETAILED DESCRIPTION OF THE INVENTION

An engineered human (h) ornithine transcarbamylase (OTC) cDNA isprovided herein, which was designed to maximize translation and improvemRNA stability as compared to the wild-type hOTC DNA and/or mRNA. Alsoprovided herein are engineered hOTC mRNA sequences. These compositionsmay be used in therapeutic and/or prophylactic methods as describedherein. Optionally, these compositions are used in combination othertherapies consistent with the standard of care for the conditions forwhich the subject (e.g., a human subject) has been diagnosed.

For comparison purposes, a wild-type human OTC cDNA sequence isillustrated in FIG. 1A. This sequence encodes the human ornithinetranscarbamylase of the amino acid sequence of FIGS. 1A-1C. This sameamino acid sequence is encoded by the engineered hOTC genes of FIG.2A-FIG. 5. The hOTC enzyme, which may be referred to as hOTCase todistinguish from the gene, is expressed from this sequence in the formof a pre-protein having a 32 amino acid leader peptide at its N-terminus(encoded by nt 1-96 of FIG. 1, about amino acids 1 to about 32 of SEQ IDNO: 2) which is cleaved after directing the enzyme to the cellularmitochondria, leaving the 322 amino acid residue “mature” protein (aboutamino acid 33 to about amino acid 354 of SEQ ID NO: 2. This “so-calledmature” hOTCase is a homo-trimeric protein with a 322 amino acid residuesequence in each polypeptide chain. Optionally, as an alternative to thewild-type sequence of SEQ ID NO:2, one may select a sequence whichincludes one or more of the naturally occurring polymorphic positionsthat are not involved in disease: F101, L111, WI193-194 of SEQ ID NO: 2(see, e.g., www.uniprot.org/uniprot/P00480).

Although all of the engineered cDNA sequences are about 77% to about 78%identical to the wt hOTC nucleic acid sequence of FIG. 1A (SEQ ID NO:1), there are structural differences between these sequences (seealignment in FIG. 5 illustrating same). Particularly, there is about 4%difference in nucleic acid sequences between hOTCco of FIG. 2 (SEQ IDNO: 3) and the hOTCcoLW4 of FIG. 4 (SEQ ID NO: 5). There is only one ntdifference between LW-3 (FIG. 3, SEQ ID NO: 4) and LW-4 (FIG. 4, SEQ IDNO: 5), i.e., 0.094% (1/1062) difference (an A in LW-3 is changed to aGin LW-4 as shown in FIG. 5).

In one embodiment, a modified hOTC coding sequence is provided whichsequence has less than about 80% identity, preferably about 77% identityor less to the full-length wild-type hOTC coding sequence (FIG. 1A, SEQID NO: 1), which encodes functional hOTCase. In one embodiment, themodified hOTC coding sequence is characterized by improved stability ascompared to wt hOTC following AAV-mediated delivery (e.g., rAAV).Additionally or alternatively, a modified hOTC coding sequence isprovided which lacks alternative reading frames for proteins of at leastabout 9 amino acids in length. Additionally, or alternatively, amodified hOTC coding sequence is provided which has hOTCase expressionlevels at least about 25-fold, at least about 50-fold, or at least about100-fold when measured following expression from a viral vector, ascompared to the hOTCase wild-type. Additionally, or alternatively, amodified hOTC coding sequence is provided which has hOTCase liveractivity which is at least about 10-fold higher, at least about 20-foldhigher, or at least about 30-fold higher as compared to the hOTCasewild-type expressed from a viral vector.

In one embodiment, a modified hOTC coding sequence is 96% to 99.9%identical to the sequence encoding the mature enzyme (about nt 99 toabout 1068) or full-length of FIG. 4 (hOTCco-LW4, SEQ ID NO: 5), or96.5% to 99% identical, or about 97% , or about 98% identical to SEQ IDNO: 5 (FIG. 4).

In one embodiment, a modified hOTC coding sequence is 96% to 99.9%identical to the sequence encoding the mature enzyme (about nt 99 toabout 1068) of FIG. 3 (hOTCco-LW3, SEQ ID NO: 4), or 96.5% to 99%identical, or about 97% , or about 98% identical to SEQ ID NO: 4 (FIG.3).

In another embodiment, a modified hOTC coding sequence is 96% to 99.9%identical to the sequence encoding the mature enzyme (about nt 99 toabout 1068) or the full-length of FIG. 2 (hOTCco, SEQ ID NO: 3), or96.5% to 99% identical, or about 97% , or 98% identical to SEQ ID NO: 3(FIG. 2).

In still another embodiment, a modified hOTC coding sequence has thesequence encoding the mature protein (about nt 99 to about 1068) or thefull-length of hOTCco-LW5 [SEQ ID NO: 8] or hOTCco-LW6 [SEQ ID NO:9], ora sequence 96% to 99.9% identical thereto. hOTCco-LW5 and hOTCco-LW6 areabout 97% identical to each other, and each is about 78% identical tothe wild-type sequence [SEQ ID NO: 1].

The sequences of FIGS. 2-5 are provided as the sense strand of the cDNAsequences. The present invention also encompasses the anti-sense strandscorresponding to these cDNA sequences and corresponding RNA, e.g., mRNA,sequences. For example, the engineered mRNA of SEQ ID NO: 10,corresponds to the DNA of SEQ ID NO:4; the engineered RNA of SEQ ID NO:11, corresponds to the DNA of SEQ ID NO: 5; the engineered RNA of SEQ IDNO: 12, corresponds to the DNA of SEQ ID NO: 8; and the RNA of SEQ IDNO: 13 corresponds to the DNA of SEQ ID NO:9. These RNA sequences, andsequences which are 95% to 99%, or about 97%, or about 98% identical toone or more of these sequences are encompassed within the scope of thisinvention. Methods for aligning and determining RNA identity are knownin the art and include published and publicly available web-based orcommercially available databases and services. See, e.g., LocARNA,CARNA, as well as other programs identified elsewhere therein.

In one embodiment of the invention, the mRNA sequence may be deliveredusing a selected RNA delivery system, examples of which are suppliedherein.

Also encompassed herein are fragments, e.g., the sequences encoding thetransit peptide (amino acids 1 to about 32), about amino acids 332 toabout 354, an intermediate hOTC enzyme, or the mature enzyme, or otherfragments as may be desired. Reference may be made to SEQ ID NO:2. See,e.g., Ye et al. 2001, Hum Gene Ther 12: 1035-1046.

In another embodiment, a chimeric OTC is provided in which theN-terminal presequence of wild-type OTC is replaced with a transitsequence from another source which is compatible with the subject'ssystem such that it effectively transports the mature hOTCase encoded bythe chimeric OTC gene to the desired organelle. See, e.g., Ye et al.2001, Hum Gene Ther. 12: 1035-1046. Such transit sequences encode atransit peptide (also termed a signal peptide, targeting signal, orlocalization signal) which is fused to the coding sequence for themature hOTC of SEQ ID NO: 1, 3, 4, 5, 8 and/or 9. For example, thewild-type hOTC transit sequence corresponds to about the first 98nucleotides of SEQ ID NO: 1. To construct a chimeric OTC, thesewild-type N-terminal sequences may be removed (about nucleic acids 1 toabout nt 96-nt 98) and replaced with a heterologous transit sequence.Suitable transit peptides are preferably, although not necessarily ofhuman origin. Suitable transit peptides may be chosen fromproline.bic.nus.edu.sg/spdb/zhang270.htm, which is incorporated byreference herein, or may be determined using a variety of computationalprograms for determining the transit peptide in a selected protein.Although not limited, such sequences may be from about 15 to about 50amino acids in length, or about 20 to about 28 amino acids in length, ormay be larger or smaller as required.

The term “percent (%) identity”, “sequence identity”, “percent sequenceidentity”, or “percent identical” in the context of nucleic acidsequences refers to the residues in the two sequences which are the samewhen aligned for correspondence. The length of sequence identitycomparison may be over the full-length of the genome, the full-length ofa gene coding sequence, or a fragment of at least about 500 to 5000nucleotides, is desired. However, identity among smaller fragments, e.g.of at least about nine nucleotides, usually at least about 20 to 24nucleotides, at least about 28 to 32 nucleotides, at least about 36 ormore nucleotides, may also be desired.

Percent identity may be readily determined for amino acid sequences overthe full-length of a protein, polypeptide, about 32 amino acids, about330 amino acids, or a peptide fragment thereof or the correspondingnucleic acid sequence coding sequences. A suitable amino acid fragmentmay be at least about 8 amino acids in length, and may be up to about700 amino acids. Generally, when referring to “identity”, “homology”, or“similarity” between two different sequences, “identity”, “homology” or“similarity” is determined in reference to “aligned” sequences.“Aligned” sequences or “alignments” refer to multiple nucleic acidsequences or protein (amino acids) sequences, often containingcorrections for missing or additional bases or amino acids as comparedto a reference sequence.

Alignments are performed using any of a variety of publicly orcommercially available Multiple Sequence Alignment Programs. Sequencealignment programs are available for amino acid sequences, e.g., the“Clustal X”, “MAP”, “PIMA”, “MSA”, “BLOCKMAKER”, “MEME”, and “Match-Box”programs. Generally, any of these programs are used at default settings,although one of skill in the art can alter these settings as needed.Alternatively, one of skill in the art can utilize another algorithm orcomputer program which provides at least the level of identity oralignment as that provided by the referenced algorithms and programs.See, e.g., J. D. Thomson et al, Nucl. Acids. Res., “A comprehensivecomparison of multiple sequence alignments”, 27(13):2682-2690 (1999).

Multiple sequence alignment programs are also available for nucleic acidsequences. Examples of such programs include, “Clustal W”, “CAP SequenceAssembly”, “BLAST”, “MAP”, and “MEME”, which are accessible through WebServers on the internet. Other sources for such programs are known tothose of skill in the art. Alternatively, Vector NTI utilities are alsoused. There are also a number of algorithms known in the art that can beused to measure nucleotide sequence identity, including those containedin the programs described above. As another example, polynucleotidesequences can be compared using Fasta™, a program in GCG Version 6.1.Fasta™ provides alignments and percent sequence identity of the regionsof the best overlap between the query and search sequences. Forinstance, percent sequence identity between nucleic acid sequences canbe determined using Fasta™ with its default parameters (a word size of 6and the NOPAM factor for the scoring matrix) as provided in GCG Version6.1, herein incorporated by reference.

In one embodiment, the modified hOTC genes described herein areengineered into a suitable genetic element (vector) useful forgenerating viral vectors and/or for delivery to a host cell, e.g., nakedDNA, phage, transposon, cosmid, episome, etc., which transfers the hOTCsequences carried thereon. The selected vector may be delivered by anysuitable method, including transfection, electroporation, liposomedelivery, membrane fusion techniques, high velocity DNA-coated pellets,viral infection and protoplast fusion. The methods used to make suchconstructs are known to those with skill in nucleic acid manipulationand include genetic engineering, recombinant engineering, and synthetictechniques. See, e.g., Sambrook et al, Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.

As used herein, an “expression cassette” refers to a nucleic acidmolecule which comprises the hOTC sequences, promoter, and may includeother regulatory sequences therefor, which cassette may be packaged intothe capsid of a viral vector (e.g., a viral particle). Typically, suchan expression cassette for generating a viral vector contains the hOTCsequences described herein flanked by packaging signals of the viralgenome and other expression control sequences such as those describedherein. For example, for an AAV viral vector, the packaging signals arethe 5′ inverted terminal repeat (ITR) and the 3′ ITR.

In one embodiment, the ITR sequences from AAV2, or the deleted versionthereof (ΔITR), are used for convenience and to accelerate regulatoryapproval. However, ITRs from other AAV sources may be selected. Wherethe source of the ITRs is from AAV2 and the AAV capsid is from anotherAAV source, the resulting vector may be termed pseudotyped.

Typically, an expression cassette for an AAV vector comprises an AAV 5′ITR, the hOTC coding sequences and any regulatory sequences, and an AAV3′ ITR. However, other configurations of these elements may be suitable.A shortened version of the 5′ ITR, termed ΔITR, has been described inwhich the D-sequence and terminal resolution site (trs) are deleted. Inother embodiments, the full-length AAV 5′ and 3′ ITRs are used.

In one embodiment, the construct is a DNA molecule (e.g., a plasmid)useful for generating viral vectors. An illustrative plasmid containingdesirable vector elements is illustrated by pAAVsc.TBG.hOTCco-LW4, thesequence of which is SEQ ID NO: 6 and which is incorporated byreference. This illustrative plasmid contains an expression cassettecomprising: scITR (nt 5-109 of SEQ ID NO: 6), a TATA signal (nt 851-854of SEQ ID NO:6), a synthetic hOTC coding sequence (nt 976-2037 of SEQ IDNO: 6), a poly A (nt 2182-2046 on the complement of SEQ ID NO: 6), ascITR (nt 2378-2211on the complement of SEQ ID NO: 6), and a liverspecific (TBG) promoter (nt 4172-4760) of SEQ ID NO: 6). Otherexpression cassettes may be generated using the other synthetic hOTCcoding sequences, and other expression control elements, describedherein.

The abbreviation “sc” in this context refers to self-complementary.“Self-complementary AAV” refers a construct in which a coding regioncarried by a recombinant AAV nucleic acid sequence has been designed toform an intra-molecular double-stranded DNA template. Upon infection,rather than waiting for cell mediated synthesis of the second strand,the two complementary halves of scAAV will associate to form one doublestranded DNA (dsDNA) unit that is ready for immediate replication andtranscription. See, e.g., D M McCarty et al, “Self-complementaryrecombinant adeno-associated virus (scAAV) vectors promote efficienttransduction independently of DNA synthesis”, Gene Therapy, (August2001), Vol 8, Number 16, Pages 1248-1254. Self-complementary AAVs aredescribed in, e.g., U.S. Pat. Nos. 6,596,535; 7,125,717; and 7,456,683,each of which is incorporated herein by reference in its entirety.

The expression cassette typically contains a promoter sequence as partof the expression control sequences, e.g., located between the selected5′ ITR sequence and the hOTC coding sequence. The illustrative plasmidand vector described herein uses the liver-specific promoter thyroxinbinding globulin (TBG). Alternatively, other liver-specific promotersmay be used [see, e.g., The Liver Specific Gene Promoter Database, ColdSpring Harbor, rulai.schl.edu/LSPD/, such as, e.g., alpha 1 anti-trypsin(A1AT); human albumin Miyatake et al., J. Virol., 71:5124 32 (1997),humAlb; and hepatitis B virus core promoter, Sandig et at, Gene Ther.,3:1002 9 (1996)]. TTR minimal enhancer/promoter, alpha-antitrypsinpromoter, LSP (845 nt)25(requires intron-less scAAV); or LSP1. Althoughless desired, other promoters, such as constitutive promoters,regulatable promoters [see, e.g., WO 2011/126808 and WO 2013/04943], ora promoter responsive to physiologic cues may be used in the vectorsdescribed herein.

In addition to a promoter, an expression cassette and/or a vector maycontain one or more other appropriate transcription initiation,termination, enhancer sequences, efficient RNA processing signals suchas splicing and polyadenylation (polyA) signals; sequences thatstabilize cytoplasmic mRNA; sequences that enhance translationefficiency (i.e., Kozak consensus sequence); sequences that enhanceprotein stability; and when desired, sequences that enhance secretion ofthe encoded product. Examples of suitable polyA sequences include, e.g.,SV40, SV50, bovine growth hormone (bGH), human growth hormone, andsynthetic polyAs. Examples of suitable enhancers include, e.g., thealpha fetoprotein enhancer, the TTR minimal promoter/enhancer, LSP(TH-binding globulin promoter/alphal-microglobulin/bikunin enhancer),amongst others. In one embodiment, the expression cassette comprises oneor more expression enhancers. In one embodiment, the expression cassettecontains two or more expression enhancers. These enhancers may be thesame or may differ from one another. For example, an enhancer mayinclude an Alpha mic/bik enhancer. This enhancer may be present in twocopies which are located adjacent to one another. Alternatively, thedual copies of the enhancer may be separated by one or more sequences.In still another embodiment, the expression cassette further contains anintron, e.g, the Promega intron. Other suitable introns include thoseknown in the art, e.g., such as are described in WO 2011/126808.Optionally, one or more sequences may be selected to stabilize mRNA. Anexample of such a sequence is a modified WPRE sequence, which may beengineered upstream of the polyA sequence and downstream of the codingsequence [see, e.g., MA Zanta-Boussif, et al, Gene Therapy (2009) 16:605-619.

These control sequences are “operably linked” to the hOTC genesequences. As used herein, the term “operably linked” refers to bothexpression control sequences that are contiguous with the gene ofinterest and expression control sequences that act in trans or at adistance to control the gene of interest.

Recombinant AAV viral vectors are well suited for delivery of the hOTCexpression sequences described herein. Such AAV vectors may contain ITRswhich are from the same AAV source as the capsid. Alternatively, the AAVITRs may be from a different AAV source than that which supplies thecapsid.

Where pseudotyped AAV is to be produced, the ITRs in the expression areselected from a source which differs from the AAV source of the capsid.For example, AAV2 ITRs may be selected for use with an AAV capsid havinga particular efficiency for targeting liver (e.g., hepatocytes). AAVcapsids may be selected from AAV8 [U.S. Pat. Nos. 7,790,449; 7,282,199]and rh10 [WO 2003/042397] for the compositions described herein.However, other AAV, including, e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6,AAV7, AAV9, and others such as, e.g., those described in WO 2003/042397;WO 2005/033321, WO 2006/110689; U.S. Pat. No. 7,588,772 B2, which areincorporated by reference herein] may be used in human subjects.

In one embodiment, a self-complementary AAV is provided. This viralvector may contain a Δ5′ ITR and an AAV 3′ ITR. In one example, theviral vector is scAAV2/8.TBG.hOTCco. In another example, the viralvector is scAAV2/rh10.TBG.hOTCco. These vectors both contain the 5′ ΔITRfrom AAV2, the liver-specific TBG promoter, an engineered hOTCco codingsequence of the invention, an SV40 polyA, and the 3′ AAV2 ITR in an AAV8capsid [see, e.g., U.S. Pat. No. 8,318,480B2] or AAV rh10 capsid. Thesequence may be selected from engineered hOTC of one of SEQ ID NO: 3, 4,5, 8 or 9. Optionally, the transit sequence of the engineered hOTC maybe substituted with a heterologous transit sequence to provide achimeric hOTC, which retains the mature hOTCase.

In another embodiment, a single-stranded AAV viral vector is provided.Such a vector may contain a 5′ AAV ITR and a 3′ ITR. One example isAAV2/8.TBG.hOTCco, which contains the full-length AAV2-5′ ITR, theliver-specific TBG promoter, the hOTC coding sequence, a bovine growthhormone polyA, and AAV2

3′ ITR. Another example is AAV2/8.TBG.hOTCco-.WPRE.bGH, which containsthe same vector elements, and additionally contains the woodchuckhepatitis virus post-transcriptional regulatory element (WPRE). In otherembodiments, WPRE is absent from constructs to be used in vivo. Theengineered hOTC sequence (abbreviated herein hOTCco) may be selectedfrom engineered hOTC of one of SEQ ID NO: 3, 4, 5, 8 or 9. Optionally,the transit peptide sequence of the engineered hOTC may be substitutedwith a heterologous transit sequence to provide a chimeric hOTC, whichretains the mature hOTCase.

Still other promoters may be selected, including tissue specificpromoters. Methods for generating and isolating AAV viral vectorssuitable for delivery to a subject are known in the art. See, e.g. USPublished Patent Application No. 2007/0036760 (Feb. 15, 2007), U.S. Pat.Nos. 7,790,449; 7,282,199; WO 2003/042397; WO 2005/033321, WO2006/110689; and U.S. Pat. No. 7,588,772 B2]. The sequences of AAV8 andmethods of generating vectors based on the AAV8 capsid are described inU.S. Pat. Nos. 7,282,199 B2, 7,790,449, and 8,318,480, which areincorporated herein by reference. In a one system, a producer cell lineis transiently transfected with a construct that encodes the transgeneflanked by ITRs and a construct(s) that encodes rep and cap. In a secondsystem, a packaging cell line that stably supplies rep and cap istransiently transfected with a construct encoding the transgene flankedby ITRs. In each of these systems, AAV virions are produced in responseto infection with helper adenovirus or herpesvirus, requiring theseparation of the rAAVs from contaminating virus. More recently, systemshave been developed that do not require infection with helper virus torecover the AAV—the required helper functions (i.e., adenovirus E1, E2a,VA, and E4 or herpesvirus UL5, UL8, UL52, and UL29, and herpesviruspolymerase) are also supplied, in trans, by the system. In these newersystems, the helper functions can be supplied by transient transfectionof the cells with constructs that encode the required helper functions,or the cells can be engineered to stably contain genes encoding thehelper functions, the expression of which can be controlled at thetranscriptional or posttranscriptional level. In yet another system, thetransgene flanked by ITRs and rep/cap genes are introduced into insectcells by infection with baculovirus-based vectors. For reviews on theseproduction systems, see generally, e.g., Zhang et al., 2009,“Adenovirus-adeno-associated virus hybrid for large-scale recombinantadeno-associated virus production,” Human Gene Therapy 20:922-929, thecontents of each of which is incorporated herein by reference in itsentirety. Methods of making and using these and other AAV productionsystems are also described in the following U.S. patents, the contentsof each of which is incorporated herein by reference in its entirety:U.S. Pat. No. 5,139,941; 5,741,683; 6,057,152; 6,204,059; 6,268,213;6,491,907; 6,660,514; 6,951,753; 7,094,604; 7,172,893; 7,201,898;7,229,823; and 7,439,065.

The available space for packaging may be conserved by combining morethan one transcription unit into a single construct, thus reducing theamount of required regulatory sequence space. For example, a singlepromoter may direct expression of a single RNA that encodes two or threeor more genes, and translation of the downstream genes are driven byIRES sequences. In another example, a single promoter may directexpression of an RNA that contains, in a single open reading frame(ORF), two or three or more genes separated from one another bysequences encoding a self-cleavage peptide (e.g., T2A) or a proteaserecognition site (e.g., furin). The ORF thus encodes a singlepolyprotein, which, either during (in the case of T2A) or aftertranslation, is cleaved into the individual proteins (such as, e.g.,transgene and dimerizable transcription factor). It should be noted,however, that although these IRES and polyprotein systems can be used tosave AAV packaging space, they can only be used for expression ofcomponents that can be driven by the same promoter. In anotheralternative, the transgene capacity of AAV can be increased by providingAAV ITRs of two genomes that can anneal to form head to tailconcatamers.

Optionally, the hOTC genes described herein may be used to generateviral vectors other than rAAV. Such other viral vectors may include anyvirus suitable for gene therapy may be used, including but not limitedto adenovirus; herpes virus; lentivirus; retrovirus; etc. Suitably,where one of these other vectors is generated, it is produced as areplication-defective viral vector.

A “replication-defective virus” or “viral vector” refers to a syntheticor artificial viral particle in which an expression cassette containinga gene of interest is packaged in a viral capsid or envelope, where anyviral genomic sequences also packaged within the viral capsid orenvelope are replication-deficient; i.e., they cannot generate progenyvirions but retain the ability to infect target cells. In oneembodiment, the genome of the viral vector does not include genesencoding the enzymes required to replicate (the genome can be engineeredto be “gutless”—containing only the transgene of interest flanked by thesignals required for amplification and packaging of the artificialgenome), but these genes may be supplied during production. Therefore,it is deemed safe for use in gene therapy since replication andinfection by progeny virions cannot occur except in the presence of theviral enzyme required for replication. Such replication-defectiveviruses may be adeno-associated viruses (AAV), adenoviruses,lentiviruses (integrating or non-integrating), or another suitable virussource.

The pharmaceutical compositions described herein are designed fordelivery to subjects in need thereof by any suitable route or acombination of different routes. Direct or intrahepatic delivery to theliver is desired and may optionally be performed via intravasculardelivery, e.g., via the portal vein, hepatic vein, bile duct, or bytransplant. Alternatively, other routes of administration may beselected (e.g., oral, inhalation, intranasal, intratracheal,intraarterial, intraocular, intravenous, intramuscular, and otherparenteral routes). The hOTC delivery constructs described herein may bedelivered in a single composition or multiple compositions. Optionally,two or more different AAV may be delivered [see, e.g., WO 2011/126808and WO 2013/049493]. In another embodiment, such multiple viruses maycontain different replication-defective viruses (e.g., AAV, adenovirus,and/or lentivirus). Alternatively, delivery may be mediated by non-viralconstructs, e.g., “naked DNA”, “naked plasmid DNA”, RNA, and mRNA;coupled with various delivery compositions and nano particles,including, e.g., micelles, liposomes, cationic lipid-nucleic acidcompositions, poly-glycan compositions and other polymers, lipid and/orcholesterol-based-nucleic acid conjugates, and other constructs such asare described herein. See, e.g., X. Su et al, Mol. Pharmaceutics, 2011,8 (3), pp 774-787; web publication: Mar. 21, 2011; WO2013/182683, WO2010/053572 and WO 2012/170930, both of which are incorporated herein byreference, Such non-viral hOTC delivery constructs may be administeredby the routes described previously.

The viral vectors, or non-viral DNA or RNA transfer moieties, can beformulated with a physiologically acceptable carrier for use in genetransfer and gene therapy applications. In the case of AAV viralvectors, quantification of the genome copies (“GC”) may be used as themeasure of the dose contained in the formulation. Any method known inthe art can be used to determine the genome copy (GC) number of thereplication-defective virus compositions of the invention. One methodfor performing AAV GC number titration is as follows: Purified AAVvector samples are first treated with DNase to eliminate un-encapsidatedAAV genome DNA or contaminating plasmid DNA from the production process.The DNase resistant particles are then subjected to heat treatment torelease the genome from the capsid. The released genomes are thenquantitated by real-time PCR using primer/probe sets targeting specificregion of the viral genome (usually poly A signal). Thereplication-defective virus compositions can be formulated in dosageunits to contain an amount of replication-defective virus that is in therange of about 1.0×10⁹ GC to about 1.0×10¹⁵ GC (to treat an averagesubject of 70 kg in body weight), and preferably 1.0×10¹² GC to 1.0×10¹⁴GC for a human patient. Preferably, the dose of replication-defectivevirus in the formulation is 1.0×10⁹ GC, 5.0×10⁹ GC, 1.0×10¹⁰ GC,5.0×10¹⁰ GC, 1.0×10¹¹ GC, 5.0×10¹¹ GC, 1.0×10¹² GC, 5.0×10¹² GC, or1.0×10¹³ GC, 5.0×10¹³ GC, 1.0×10¹⁴ GC, 5.0×10¹⁴ GC, or 1.0×10¹⁵ GC.

DNA and RNA is generally measured in the nanogram (ng) to microgram (μg)amounts of the nucleic acids. In general, for a treatment in a humanpreferably dosages of the RNA is the range of 1 ng to 700 μg, 1 ng to500 μg, 1 ng to 300 μg, 1 ng to 200 μg, or 1 ng to 100 μg are formulatedand administered. Similar dosage amounts of a DNA molecule containing anexpression cassette and not delivered to a subject via a viral vectormay be utilized for non-viral hOTC DNA delivery constructs.

Production of lentivirus is measured as described herein and expressedas IU per volume (e.g., mL). IU is infectious unit, or alternativelytransduction units (TU); IU and TU can be used interchangeably as aquantitative measure of the titer of a viral vector particlepreparation. The lentiviral vector is typically non-integrating. Theamount of viral particles is at least about 3×10⁶ IU, and can be atleast about 1×10⁷ IU, at least about 3×10⁷IU, at least about 1×10⁸ IU,at least about 3×10⁸ IU, at least about 1×10⁹ IU, or at least about3×10⁹ IU.

The above-described recombinant vectors may be delivered to host cellsaccording to published methods. The rAAV, preferably suspended in aphysiologically compatible carrier, may be administered to a human ornon-human mammalian patient. Suitable carriers may be readily selectedby one of skill in the art in view of the indication for which thetransfer virus is directed. For example, one suitable carrier includessaline, which may be formulated with a variety of buffering solutions(e.g., phosphate buffered saline). Other exemplary carriers includesterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran,agar, pectin, peanut oil, sesame oil, and water. The selection of thecarrier is not a limitation of the present invention.

Optionally, the compositions of the invention may contain, in additionto the rAAV and carrier(s), other conventional pharmaceuticalingredients, such as preservatives, or chemical stabilizers. Suitableexemplary preservatives include chlorobutanol, potassium sorbate, sorbicacid, sulfur dioxide, propyl gallate, the parabens, ethyl vanillin,glycerin, phenol, and parachlorophenol. Suitable chemical stabilizersinclude gelatin and albumin.

The viral vectors described herein may be used in preparing a medicamentfor delivering ornithine transcarbamylase to a subject (e.g., a humanpatient) in need thereof, supplying functional hOTCase to a subject,and/or for treating ornithine transcarbamylase deficiency. A course oftreatment may optionally involve repeat administration of the same viralvector (e.g., an AAV8 vector) or a different viral vector (e.g., an AAV8and an AAVrh10). Still other combinations may be selected using theviral vectors and non-viral delivery systems described herein.

In another embodiment, the nucleic acid sequences described herein maybe delivered via a non-viral route. For example, a hOTC sequence may bevia a carrier system for expression or delivery in RNA form (e.g., mRNA)using one of a number of carrier systems which are known in the art.Such carrier systems include those provided by commercial entities, suchas PhaseRx' so-called “SMARTT” technology. These systems utilize blockcopolymers for delivery to a target host cell. See, e.g., US2011/0286957 entitled, “Multiblock Polymers”, published Nov. 24, 2011;US 2011/0281354, published Nov. 17, 2011; EP2620161, published Jul. 31,2013; and WO 2015/017519, published Feb. 5, 2015,. See, also, S. Uchidaet al, (February 2013) PLoS ONE 8(2): e56220. Still other methodsinvolve generating and injecting synthetic dsRNAs [see Soutschek et al.Nature (2004) 432(7014): 173-8; see also Morrissey et al Hepatol. (2005)41(6): 1349-56], local administration to the liver has also beendemonstrated by injecting double stranded RNA directly into thecirculatory system surrounding the liver using renal veincatheterization. [See Hamar et al. PNAS (2004) 101(41): 14883-8.]. Stillother systems involve delivery of dsRNA and particularly siRNA usingcationic complexes or liposomal formulations [see, e.g., Landen et al.Cancer Biol. Ther. (2006) 5(12); see also Khoury et al. ArthritisRheumatol. (2006) 54(6): 1867-77. Other RNA delivery technologies arealso available, e.g., from Veritas Bio [see, e.g., US 2010/0323001, Dec.23, 2010, “In vivo delivery of double stranded RNA to a target cell”(cytosolic content including RNAs, e.g., mRNA, expressedsiRNA/shRNA/miRNA, as well as injected/introduced siRNA/shRNA/miRNA, orpossibly even transfected DNA present in the cytosol packaged withinexovesicles and be transported to distal sites such as the liver)].Still other systems for in vivo delivery of RNA sequences have beendescribed. See, e.g., US 2012/0195917 (Aug. 2, 2012) (5′-cap analogs ofRNA to improve stability and increase RNA expression), WO 2013/143555A1,Oct. 3, 2013, and/or are commercially available (BioNTech, Germany;Valera (Cambridge, Mass.); Zata Pharmaceuticals).

Thus, in one embodiment, the invention provides an engineered hOTC mRNAof the mature sequence (at least about nt 99-1098) or the full-length ofSEQ ID NO: 10, 11, 12, 13, or a sequence having at least 97% to 99%identity thereto, in a composition for delivery of double-stranded orsingle stranded RNA which results in expression of the mature hOTCase ina target host cell, e.g., a liver cell.

The kinetics of the composition described herein which contain mRNA(delivered directly, as compared to transcribed from a DNA deliverymolecule) are particularly well suited for use in subjects in acutecrisis, as expression of the hOTCase from the mRNA may be seen within aperiod of several hours. In order to avoid rapid clearance of the RNA,it is modified as described herein (e.g., using a cap or a modifiedbase), such that its effects may be retained for over 24 hours, over 48hours, or up to about 3 days (about 72 hours). It may be desirable toco-administer an mRNA directly as described herein and co-administer atthe same or substantially the same time, a DNA or viral vector-basedhOTC composition as defined herein. Thus, a subject may receiveimmediate treatment, and at such time as the mRNA-mediated expressionbegins to wane, the longer-term hOTC expression conferred by a viralvector-mediated expression begins to take effect. Alternatively, asubject may receive a second administration of an mRNA-based compositionas defined herein. The mRNA compositions described herein may be used inother therapeutic regimens or methods, including those involving OTCDpatients who are not in acute crisis.

The compositions according to the present invention may comprise apharmaceutically acceptable carrier, such as defined above. In oneembodiment, the composition comprises a block copolymer associated witha hOTC polynucleotide as described herein. The block copolymers may forma micelle, such that the micelle comprises a plurality of blockcopolymer.

Typically, such a composition contains a nucleic acid moleculecomprising the mRNA sequence corresponding to the hOTC sequence encodingthe mature hOTCase (at least about nt 99 to 1098) or the full-length ofany of SEQ ID NO: 10, 11, 12, 13. In addition, this nucleic acidmolecule may include the 5′ untranslated region (UTR), also known as theleader sequence or leader RNA, and one or more of an optional intron(s),an optional exon(s), an optional a Kozak sequence, an optional WPRE. anda polyA, and the 3′ UTR flanking the coding sequences. Suitable leadersequences include those discussed above in connection with the hOTC DNAsequences, which discussion is incorporated by reference herein.Examples of sources of suitable leader sequences, other than the nativehOTC leader sequences, or those corresponding to FIG. 2, FIG. 3 or FIG.4, or FIG. 5 are discussed above. Similarly, sources of suitableintrons, polyA, and Kozak sequences are discussed above and areapplicable to the delivery of the corresponding RNA sequences discussedin the present paragraph. Further, various modifications to the RNA maybe generated, e.g., a modified 5′ cap structure may be engineered intothe construct in order to avoid rapid clearance of the mRNA in vivo, orfor another desired reason. Methods of generating such 5′ cap structuresis known to those of skill in the art. See, e.g., US 2012/0195917 and WO2013/143555A1, Oct. 3, 2013. In addition, modified nucleotides can beused to make mRNA in vitro, like pseudouridine. Also RNA may be dosedrepetitively, or subject can be dosed first with mRNA to manage neonatalcrises followed up by viral vector-mediated delivery (e.g., AAV) forlong term therapy and to prevent fibrosis/cirrhosis and/orhepatocellular carcinoma.

mRNA can be synthesized from the hOTC DNA sequences described herein,using techniques that are well known in the art. For example, CazenaveC, Uhlenbeck O C, RNA template-directed RNA synthesis by T7 RNApolymerase. Proc Natl Acad Sci U S A. 1994 Jul. 19; 91(15):6972-6,describe the use of the T7 RNA polymerase for generating RNA from cDNAor RNA templates. See also, Wichlacz Al, Legiewicz M, Ciesiolka J.,Generating in vitro transcripts with homogenous 3′ ends usingtrans-acting antigenomic delta ribozyme., Nucleic Acids Res. 2004 Feb.18; 32(3):e39; Krieg P A, Melton D A., Functional messenger RNAs areproduced by SP6 in vitro transcription of cloned cDNAs, Nucleic AcidsRes. 1984 Sep. 25; 12(18):7057-70; and Rio, D. C., et al. RNA: ALaboratory Manual. Cold Spring Harbor: Cold Spring Harbor LaboratoryPress, 2011, 205-220. Each of these references is incorporated herein byreference. In addition, kits and protocols for generating mRNA areavailable commercially including, without limitation, the Riboprobe® InVitro Transcription System (Promega Corp.); RiboMAX™ Large Scale RNAProduction Systems (Promega Corp.); MAXIscript Kit (Ambion); MEGIscriptKit (Ambion); MessageAmp™ aRNA Kit (Ambion); mMESSAGE mMACHINE® Kits(Ambion); and HiScribe™ T7 High Yield RNA Synthesis Kit (New EnglandBiolabs® Inc.). Custom RNA can also be generated commercially fromcompanies including, without limitation, TriLink Biotechnologies;bioSYNTHESIS; GE Dharmacon; and IBA Lifesciences.

The hOTC DNA sequences described herein can be generated in vitro andsynthetically, using techniques well known in the art. For example, thePCR-based accurate synthesis (PAS) of long DNA sequence method may beutilized, as described by Xiong et al, PCR-based accurate synthesis oflong DNA sequences, Nature Protocols 1, 791-797 (2006). A methodcombining the dual asymmetrical PCR and overlap extension PCR methods isdescribed by Young and Dong, Two-step total gene synthesis method,Nucleic Acids Res. 2004; 32(7): e59. See also, Gordeeva et al, JMicrobiol Methods. Improved PCR-based gene synthesis method and itsapplication to the Citrobacter freundii phytase gene codon modification.2010 May; 81(2):147-52. Epub 2010 Mar. 10; see, also, the followingpatents on oligonucleotide synthesis and gene synthesis, Gene Seq. 2012April; 6(1):10-21; U.S. Pat. Nos. 8,008,005; and 7,985,565. Each ofthese documents is incorporated herein by reference. In addition, kitsand protocols for generating DNA via PCR are available commercially.These include the use of polymerases including, without limitation, Taqpolymerase; OneTaq® (New England Biolabs); Q5® High-Fidelity DNAPolymerase (New England Biolabs); and GoTaq® G2 Polymerase (Promega).DNA may also be generated from cells transfected with plasmidscontaining the hOTC sequences described herein. Kits and protocols areknown and commercially available and include, without limitation, QIAGENplasmid kits; Chargeswitch® Pro Filter Plasmid Kits (Invitrogen); andGenElute™ Plasmid Kits (Sigma Aldrich). Other techniques useful hereininclude sequence-specific isothermal amplification methods, thateliminate the need for thermocycling. Instead of heat, these methodstypically employ a strand-displacing DNA polymerase, like Bst DNAPolymerase, Large Fragment (New England Biolabs), to separate duplexDNA. DNA may also be generated from RNA molecules through amplificationvia the use of Reverse Transcriptases (RT), which are RNA-dependent DNAPolymerases. RTs polymerize a strand of DNA that is complimentary to theoriginal RNA template and is referred to as cDNA. This cDNA can then befurther amplified through PCR or isothermal methods as outlined above.Custom DNA can also be generated commercially from companies including,without limitation, GenScript; GENEWIZ®; GeneArt® (Life Technologies);and Integrated DNA Technologies.

The term “expression” is used herein in its broadest meaning andcomprises the production of RNA or of RNA and protein. With respect toRNA, the term “expression” or “translation” relates in particular to theproduction of peptides or proteins. Expression may be transient or maybe stable.

The term “translation” in the context of the present invention relatesto a process at the ribosome, wherein an mRNA strand controls theassembly of an amino acid sequence to generate a protein or a peptide.

According to the present invention, a “therapeutically effective amount”of the hOTC is delivered as described herein to achieve a desiredresult, i.e., treatment of OTC deficiency or one or more symptomsthereof. As described herein, a desired result includes reducing oroticacid levels, reducing hyperammonemia and/or minimizing or eliminatingone or more of the neurophysical complications including developmentaldelay, learning disabilities, intellectual disability, attention deficithyperactivity disorder, and executive function deficits. Treatment mayinclude treatment of subjects having severe neonatal-onset disease(males or, more rarely, females), and late-onset (partial) disease inmales and females, which may present from infancy to later childhood,adolescence, or adulthood. In certain embodiments, the inventionprovides a method of treating and/or preventing fibrosis and/orcirrhosis in subjects, particularly those late-onset heterozygoussubjects by administering hOTC as described herein. In one embodiment,therapeutic goals for OTC deficiency are to maintain plasma ammonia atless than <80 μmol/L, plasma glutamine <1,000 μmol/L, argininemia 80-150μmol/L and branched chain amino acids within the normal range. However,other therapeutic endpoints may be selected by the treating physician.

In yet another embodiment, the invention provides a method of rescuingand/or treating a neonatal subject OTCD comprising the step ofdelivering a hOTC gene to the liver of a newborn subject (e.g., a humanpatient). This method may utilize any nucleic acid sequence encoding afunctional hOTCase, whether a synthetic hOTC as described herein or awild-type hOTC, or a hOTC from another source, or a combination thereof.In one embodiment, neonatal treatment is defined as being administered ahOTC as described herein within 8 hours, the first 12 hours, the first24 hours, or the first 48 hours of delivery. In another embodiment,particularly for a primate, neonatal delivery is within the period ofabout 12 hours to about 1 week, 2 weeks, 3 weeks, or about 1 month, orafter about 24 hours to about 48 hours. To address dilution due to therapid turnover of liver cells in a growing mammal (e.g., a non-human orhuman primate), neonatal therapy is desirably followed byreadministration at about 3 months of age, about 6 months, about 9months, or about 12 months. Optionally, more than one readministrationis permitted. Such readministration may be with the same type of vector,a different viral vector, or via non-viral delivery. In one embodiment,an RNA based delivery system for functional hOTC is used to stabilize asubject (e.g., a human patient) in crisis, followed by delivery of aviral vector mediated delivery of a functional hOTC. In anotherembodiment, initial therapy involves co-administration of viral andnon-viral-mediated hOTC delivery systems. In a further embodiment, thehOTC DNA and RNA constructs may be used alone, or in combination withthe standard of care for the patient's diagnosis and condition.

As described in the working examples herein, the inventors have foundthat heterozygous OTCD subjects, including those with late onset OTCD,have increased fibrosis and/or microvesicular steatosis throughout theliver. Such liver fibrosis and/or microvesicular steatosis can lead toOTCD-related cirrhosis. Thus, in another embodiment, the inventionprovides methods of preventing liver fibrosis and/or the associatedmedical condition OTCD-related cirrhosis by delivering to the subject(e.g., a human patient) a hOTC. This aspect of the invention may utilizea viral or non-viral delivery system. The nucleic acid expressioncassette may contain a synthetic hOTC DNA or RNA as provided herein, oranother suitable sequence which expresses functional hOTCase. In oneembodiment, a method of treating and/or preventing liver fibrosis,microvesicular steatosis, and/or OTCD-related cirrhosis is providedwhich involves delivering OTCase to a subject having OTCD. The subjectmay be a human patient. In one embodiment, the patient is heterozygousand has late onset OTCD. The patient may have been previously untreatedfor OTCD, or may have received other conventional treatments. Atpresent, there is no existing standard of care for OTCD, but rathersymptoms are managed, e.g., through discontinuation of protein intake,compensatory increases in dietary carbohydrates and lipids, hemodialysisfor comatose patients with extremely high blood levels; and/orintravenous administration of sodium benzoate, arginine, and/or sodiumphenylacetate. The US FDA has approved glycerol phenylbutyrate(Ravicti®) for long-term management of some urea cycle disorders forpatients aged 2 years and older; this drug helps rid the body of ammoniaand is intended for patients who cannot be managed by aprotein-restricted diet or amino acid supplements alone. In oneembodiment, treatment of the patient (e.g., a first injection) isinitiated prior to the first year of life. In another embodiment,treatment is initiated after the first 1 year, or after the first 2 to 3years of age, after 5 years of age, after 11 years of age, or at anolder age.

In one embodiment, the method of the invention provides for treatingand/or reversing liver fibrosis and/or OTCD-related cirrhosis bydelivering to the subject a functional OTCase which is encoded by anengineered DNA of SEQ ID NO: 1, 3, 4, 5, 8 or 9, or a chimeric DNA asdefined herein. Delivery of the DNA may be mediated by a viral vectorcontaining the engineered DNA in an expression cassette, or by anon-viral delivery system, either of which mediates expression offunctional OTCase in the liver cells of the subject. In anotherembodiment, the subject is administered an engineered RNA of SEQ ID NO:10, 11, 12 or 13, or a chimeric RNA as defined herein. Delivery of theRNA may be mediated by a viral vector containing the engineered RNA inan expression cassette, or by a non-viral delivery system, either ofwhich mediates expression of functional OTCase in the liver cells of thesubject.

Heterozygous OTCD subjects have an increased risk of developinghepatocellular carcinoma (HCC). See, e.g., J M Wilson et al, MoleuclarGenetics and Metabolism (2012), Mol Genet Metab. 2012 February; 105(2):263-265, Published online 2011 Nov. 7. Thus, in another embodiment, theinvention provides methods of preventing treating and/or preventing HCCby delivering to the subject (e.g., a human patient) a hOTC. This aspectof the invention may utilize a viral or non-viral delivery system. Thenucleic acid expression cassette may contain a synthetic hOTC DNA or RNAas provided herein, or another suitable sequence which expressesfunctional hOTCase. The patient may have been previously untreated forOTCD, or may have received other conventional treatments, i.e., standardof care. In one embodiment, treatment of the patient (e.g., a firstinjection) is initiated prior diagnosis with HCC. In another embodiment,treatment of the patient is initiated following HCC diagnosis.Optionally, treatment involves co-administration with sorafenib(commercially available as Nexavar®), or being used in conjunction withchemoembolization, radiation, thermal ablation, percutanous ethanolinjection, targeted therapy (e.g., anti-angiogenesis drugs), hepaticarterial infusion of anti-cancer drugs, immunotherapy, or with surgicaloptions including, e.g., resection, cryosurgery, and liver transplant.When used for treatment of HCC, it may be desirable to select anon-integrating delivery system (e.g., direct RNA delivery, ornon-integrating viruses such as adenoviruses or non-integratinglentiviruses) for delivery of a synthetic hOTC DNA or RNA as describedherein.

By “functional OTC”, is meant a gene which encodes the wild-type OTCasesuch as characterized by SEQ ID NO: 2 or another OTCase which providesat least about 50%, at least about 75%, at least about 80%, at leastabout 90%, or about the same, or greater than 100% of the biologicalactivity level of the wild-type human ornithine transcarbamylase enzyme,which may be characterized by the sequence of SEQ ID NO:2 or a naturalvariant or polymorph thereof which is not associated with disease. Moreparticularly, as heterozygous patients may have as low an OTCasefunctional level as about 50% or lower, effective treatment may notrequire replacement of OTCase activity to levels within the range of“normal” or non-OTCD patients. Similarly, patients having no detectableamounts of OTCase may be rescued by delivering OTCase function to lessthan 100% activity levels, and may optionally be subject to furthertreatment subsequently. As described herein, the gene therapy describedherein, whether viral or non-viral, may be used in conjunction withother treatments, i.e., the standard of care for the subject's(patient's) diagnosis.

In one embodiment, such a functional OTCase has a sequence which hasabout 95% or greater identity to the mature protein (i.e., about thelast 322 amino acids) or full-length sequence of SEQ ID NO: 2, or about97% identity or greater, or about 99% or greater to SEQ ID NO: 2 at theamino acid level. Such a functional OTCase may also encompass naturalpolymorphs which are not associated with any disease (e.g., F101, L111,and/or WI193-194 of SEQ ID NO: 2). Identity may be determined bypreparing an alignment of the sequences and through the use of a varietyof algorithms and/or computer programs known in the art or commerciallyavailable [e.g., BLAST, ExPASy; ClustalO; FASTA; using, e.g.,Needleman-Wunsch algorithm, Smith-Waterman algorithm].

A variety of assays exist for measuring OTC expression and activitylevels in vitro. See, e.g., X Ye, et al, 1996 Prolonged metaboliccorrection in adult ornithine transcarbamylase-deficient mice withadenoviral vectors. J Biol Chem 271:3639-3646) or in vivo. For example,OTC enzyme activity can be measured using a liquid chromatography massspectrometry stable isotope dilution method to detect the formation ofcitrulline normalized to [1,2,3,4,5-13C5] citrulline (98% 13C). Themethod is adapted from a previously developed assay for detection ofN-acetylglutamate synthase activity [Morizono H, et al, MammalianN-acetylglutamate synthase. Mol Genet Metab. 2004; 81(Suppl 1):54-11.].Slivers of fresh frozen liver are weighed and briefly homogenized inbuffer containing 10 mM HEPES, 0.5% Triton X-100, 2.0 mM EDTA and 0.5 mMDTT. Volume of homogenization buffer is adjusted to obtain 50 mg/mltissue. Enzyme activity is measured using 250 μg liver tissue in 50 mMTris-acetate, 4 mM ornithine, 5 mM carbamyl phosphate, pH 8.3. Enzymeactivity is initiated with the addition of freshly prepared 50 mMcarbamyl phosphate dissolved in 50 mM Tris-acetate pH 8.3, allowed toproceed for 5 minutes at 25° C. and quenched by addition of an equalvolume of 5 mM13C5-citrulline in 30% TCA. Debris is separated by 5minutes of microcentrifugation, and the supernatants are transferred tovials for mass spectroscopy. Ten μL of sample are injected into anAgilent 1100 series LC-MS under isocratic conditions with a mobile phaseof 93% solvent A (1 ml trifluoroacetic acid in 1 L water):7% solvent B(1 ml trifluoroacetic acid in 1 L of 1:9 water/acetonitrile). Peakscorresponding to citrulline [176.1 mass:charge ratio (m/z)] and13C5-citrulline (181.1 m/z) are quantitated, and their ratios arecompared to ratios obtained for a standard curve of citrulline run witheach assay. Samples are normalized to either total liver tissue or toprotein concentration determined using a Bio-Rad protein assay kit(Bio-Rad, Hercules, Calif.). Other assays, which do not require liverbiopsy, may also be used. One such assay is a plasma amino acid assaysin which the ratio of glutamine and citrulline is assessed and ifglutamine is high (>800 microliters/liter) and citrulline is low (e.g.,single digits), a urea cycle defect is suspected. Plasma ammonia levelscan be measured and a concentration of about 100 micromoles per liter isindicative of OTCD. Blood gases can be assessed if a patient ishyperventilating; respiratory alkalosis is frequent in OTCD. Orotic acidin urine, e.g., greater than about 20 micromoles per millimole creatineis indicative of OTCD, as is elevated urinary orotate after allopurinolchallenge test. Diagnostic criteria for OTCD have been set forth inTuchman et al, 2008, Urea Cycle Disorders Consortium (UCDC) of the RareDisease Clinical Research Network (RDCRN). Tuchman M, et al., Consortiumof the Rare Diseases Clinical Research Network. Cross-sectionalmulticenter study of patients with urea cycle disorders in the UnitedStates. Mol Genet Metab. 2008; 94:397-402, which is incorporated byreference herein. See, also, www.ncbi.nlm.nih.gov/books/NBK154378/,which provides a discussion of the present standard of care for OTCD.

It is to be noted that the term “a” or “an” refers to one or more. Assuch, the terms “a” (or “an”), “one or more,” and “at least one” areused interchangeably herein.

The words “comprise”, “comprises”, and “comprising” are to beinterpreted inclusively rather than exclusively. The words “consist”,“consisting”, and its variants, are to be interpreted exclusively,rather than inclusively. While various embodiments in the specificationare presented using “comprising” language, under other circumstances, arelated embodiment is also intended to be interpreted and describedusing “consisting of” or “consisting essentially of” language.

As used herein, the term “about” means a variability of 10% (±10%) fromthe reference given, unless otherwise specified.

The term “regulation” or variations thereof as used herein refers to theability of a compound of formula (I) to inhibit one or more componentsof a biological pathway.

A “subject” is a mammal, e.g., a human, mouse, rat, guinea pig, dog,cat, horse, cow, pig, or non-human primate, such as a monkey,chimpanzee, baboon or gorilla.

As used herein, “disease”, “disorder” and “condition” are usedinterchangeably, to indicate an abnormal state in a subject.

Unless defined otherwise in this specification, technical and scientificterms used herein have the same meaning as commonly understood by one ofordinary skill in the art and by reference to published texts, whichprovide one skilled in the art with a general guide to many of the termsused in the present application.

The following examples are illustrative only and are not intended tolimit the present invention.

EXAMPLE 1 scAAV Vectors Containing hOTC

pAAVsc.TBG.hOTCwt and pAAVsc.TBG.hOTCco-LW4 were constructed byreplacing the mOTC coding sequencing with wild-type (WT) hOTC (hOTCwt)or hOTCcoLW cDNA, respectively, in a plasmid derived from the previouslydescribed pAAVsc.TBG.mOTC1.3 with the intron disrupted [Moscioni D, etal, “Long-term correction of ammonia metabolism and prolonged survivalin ornithine transcarbamylase-deficient mice following liver-directedtreatment with adeno-associated viral vectors”, Mol Ther. 2006;14:25-33; Cunningham S.C., et al, “AAV2/8-mediated correction of OTCdeficiency is robust in adult but not neonatal Spf(ash) mice”, Mol Ther.2009; 17:1340-1346; Wang L, et al., “Sustained correction of OTCdeficiency in spf^(ash) mice using optimized self-complementary AAV2/8vectors”, Gene Ther. 2012 April; 19(4):404-10, Epub 2011 Aug. 18].

The scAAV2/8.TBG.hOTCco-LW4 contains an AAV2 3′ ITR and a 5′ ITR with adeletion in the D-sequence and trs (terminal resolution site), a TBGpromoter, the hOTCco-LW4 gene, and a 137 bp SV40 polyA. The two vectorpreps (AAV2/8sc.TBG.hOTCwt and AAV2/8sc.TBG.hOTCco-LW4) used in theinitial comparison study were purified by two rounds of cesium chloridegradient centrifugation, as previously described [Wang L, et al,Systematic evaluation of AAV vectors for liver directed gene transfer inmurine models. Mol Ther. 2010; 18:118-125]. Vectors used in the rest ofthe study were produced by a scaled production method based onpolyethylenimine (PEI) transfection and purified from supernatant ortotal lysate by iodixanol gradient centrifugation as described [Lock M,et al, Hum Gene Ther. 2010; 21:1259-1271]. Genome titers [genome copies(GC)/ml] of AAV vectors were determined by real-time PCR using primerand probe sets targeting the TBG promoter (forward primer5′-AAACTGCCAATTCCACTGCTG-3′ [SEQ ID NO: 14], reverse primer5′-CCATAGGCAAAAGCACCAAGA-3′ [SEQ ID NO: 15], probe6FAM-TTGGCCCAATAGTGAGAACTTTTTCCTGC [SEQ ID NO: 16]-TAMRA), and using alinearized plasmid as the standard. The forward primer is located 400 bpdownstream of the 5′ closed hairpin. Fagone et al [Systemic errors inquantitative polymerase chain reaction titration of self-complementaryadeno-associated viral vectors and improved over alternative methods,Hum Gene Ther Methods. 2012 February; 23(1):1-7.] recently reported thatthe quantitative PCR (Q-PCR) method could significantly underestimatethe titer of scAAV vectors, especially when the PCR primers were closeto the closed hairpin of the scAAV vector. The titer of one lot ofAAV2/8sc.TBG.hOTCco-LW4 vector using a primer and probe set targetingthe polyA region (1900 bp downstream of the 5′ closed hairpin), and thegenome titer was 1.1-fold of the original titer, which was within theintra-assay error of Q-PCR.

OTC protein expression levels and OTC activity were evaluated in theliver of spf^(ash) mice 14 days after i.v. injection of 1×10¹¹ GC ofAAV2/8sc.TBG.hOTCwt or AAV2/8sc.TBG.hOTCco-LW4 vectors. The spf^(ash)mice are a model for late onset OTC disease in humans. All animalprocedures were performed in accordance with protocols approved by theInstitutional Animal Care and Use Committee (IACUC) of the University ofPennsylvania. Spf^(ash) mice were maintained at the Animal Facility ofthe Translational Research Laboratories at the University ofPennsylvania as described previously. Three to six months old spf^(ash)mice and their normal littermates were used in the studies. Vectors wereadministered by intravenous (i.v.) injection via the tail vein. Theextent of gene transfer based on resident vector genomes was notstatistically different between the two groups. Urine samples werecollected before and at various time points after vector treatment fororotic acid analysis as previously described [Moscioni D, et al,Long-term correction of ammonia metabolism and prolonged survival inornithine transcarbamylase-deficient mice following liver-directedtreatment with adeno-associated viral vectors. Mol Ther. 2006;14:25-33].

Western blot analysis to detect hOTC expression in liver lysate wasperformed as previously described [Wang L, et al, 2012, epub 2011]. Theprimary antibody to detect hOTC was a custom rabbit polyclonal antibodyprovided by Hiroki Morizono's laboratory at the Children's NationalMedical Center. Liver lysates (10 μg/lane) were also blotted and probedby an anti-tubulin antibody (Abcam, Cambridge, Mass.). Western analysisdemonstrated 100-fold higher expression of hOTC from the hOTCco-LW4vector as compared to the hOTCwt vector, reaching levels in slightexcess of those seen in WT mice.

OTC enzyme activity was measured using a liquid chromatography massspectrometry stable isotope dilution method to detect the formation ofcitrulline normalized to [1,2,3,4,5-13C5] citrulline (98% 13C). Themethod is adapted from a previously developed assay for detection ofN-acetylglutamate synthase activity [Morizono H, et al, MammalianN-acetylglutamate synthase. Mol Genet Metab. 2004; 81(Suppl 1):S4-11.].Slivers of fresh frozen liver were weighed and briefly homogenized inbuffer containing 10 mM HEPES, 0.5% Triton X-100, 2.0 mM EDTA and 0.5 mMDTT. Volume of homogenization buffer was adjusted to obtain 50 mg/mltissue. Enzyme activity was measured using 250 μg liver tissue in 50 mMTris-acetate, 4 mM ornithine, 5 mM carbamyl phosphate, pH 8.3. Enzymeactivity was initiated with the addition of freshly prepared 50 mMcarbamyl phosphate dissolved in 50 mM Tris-acetate pH 8.3, allowed toproceed for 5 minutes at 25° C. and quenched by addition of an equalvolume of 5 mM13C5-citrulline in 30% TCA. Debris was separated by 5minutes of microcentrifugation, and the supernatants were transferred tovials for mass spectroscopy. Ten μL of sample was injected into anAgilent 1100 series LC-MS under isocratic conditions with a mobile phaseof 93% solvent A (1 ml trifluoroacetic acid in 1 L water):7% solvent B(1 ml trifluoroacetic acid in 1 L of 1:9 water/acetonitrile). Peakscorresponding to citrulline [176.1 mass:charge ratio (m/z)] and13C5-citrulline (181.1 m/z) were quantitated, and their ratios werecompared to ratios obtained for a standard curve of citrulline run witheach assay. Samples were normalized to either total liver tissue or toprotein concentration determined using a Bio-Rad protein assay kit(Bio-Rad, Hercules, Calif.).

The vector carrying engineered hOTC cDNA termed herein LW4 (FIG. 4) wasfound to improve expressed hOTC protein levels by 100-fold. Anassessment of OTC enzyme activity generally correlated with the OTCWestern blot experiments although OTC protein was more elevated than OTCenzyme activity when compared to endogenous OTC. When subtracting thebackground activity levels in the spf^(ash) mice, the hOTCco-LW4resulted in over 33-fold higher activity than the hOTCwt. Sustained anddose-correlated hOTC expression and activity levels were observed in thetreated spf^(ash) mice. Compared to a previously described murine OTCvector which differed mainly in the cDNA, the vector carrying thehOTCco-LW4 vector was about 10-fold more potent.

The illustrative vector carrying the modified hOTCco-LW4 (FIG. 4)provided high level of transduction, as measured by OTC histologicalassays, throughout a broad range of doses. Between doses 1×10¹¹ GC and 3×10⁹ GC, transduction efficiency, as measured by histochemical staining,varied between 50-70%. At the lowest dose of 1×10⁹ GC, 40% of the liverareas were positive by OTC histochemical staining. The lack of a cleardose effect by histochemistry and immunostaining could be due to thefact that codon optimization significantly improved hOTC expression inthe transduced hepatocytes. This leads to improved sensitivity to detecttransduced cells with low vector genome copies. Transduction could besaturated with high vector doses (1×10¹¹-1 ×10¹⁰ GC), and thereforetransduction efficiency measured by in situ detection methods would notdiscriminate between low and high dose groups in contrast to OTC enzymeactivity on liver lysates measured by mass spectrometry.

A further study was performed in which neonatal expression of hOTC wasassessed in spf^(ash) mice, injected on day 1 of life, using thescAAV2/8.TBG.hOTCco at a dose of 5×10¹⁰ GC/pup injected via the temporalvein. Robust expression was detected at 24 and 48 hours. Additionalstudies were performed using doses of 1×10¹¹, 3×10¹⁰, and 1×10¹⁰ , andassessed for 12 weeks. Over the 16 week period of the study, a reductionin the initial robust expression levels was observed at each of thedoses. This is believed to be due to dilution, i.e., a natural result ofthe proliferation of liver cells in growing animals. Thus, while initialrestoration of OTC liver activity is observed following neonatal genetransfer in spf^(ash) mice, this result is temporary, with OTC activitydropping from about 1000% of wild-type (wt) levels at about 1 week, toabout 50% of wt levels at 4 weeks, to about 10% of wt levels at 12 weeks(1×10¹¹ GC level); or about 500% of wt levels at week 1, to about 20% ofwt levels, or about 10% of wt levels at week 1 (3×10¹⁰ GC dose) or about200% wt levels at week 1, to about 10% wt levels at week 4 (1×10¹⁰ GCdose). In one study, using animals receiving the first injection of3×10¹⁰ GC at day 1, animals were injected with a second AAV vectorcarrying the hOTCco gene (scAAVrh10.hOTCco; 1.8×10¹⁰ GC) at week 4. As acontrol, one group of animals received no readministration and one groupreceived only the second vector at 4 weeks. Readministration of theAAV.hOTCco resulted in restoration of liver OTC activity.

Further studies were designed to assess the ability to rescue OTC-KOpups by neonatal gene therapy, both short-term and long-term.

EXAMPLE 2 Production of scAAV Vectors having Codon Optimized Sequences

A. scAAV8.TBG.hOTC-co

Plasmids containing a codon optimized hOTC sequence of SEQ ID NO: 3, 4,5, 9 or 10, respectively, are cloned as described in Example 1 byreplacing the mOTC coding sequencing with hOTCco in a plasmid derivedfrom the previously described pAAVsc.TBG.mOTC1.3 with the intron. Theresulting plasmid pAAVsc.TBG.hOTCco is cloned into an AAV8 capsid [Gaoet al, PNAS USA, 2002, 99:11854-11859] using conventional techniques.

B. scAAVrh10.TBG.hOTC-co

Plasmids containing a codon optimized hOTC sequence of SEQ ID NO: 3, 4,5, 9 or 10, respectively, are cloned as described in Example 1 byreplacing the mOTC coding sequencing with hOTCco in a plasmid derivedfrom the previously described pAAVsc.TBG.mOTC1.3 with the intron. Theresulting plasmids pAAVsc.TBG.hOTCco are cloned into a AAVrh10 capsid[Gao et al, PNAS USA, 2002, 99:11854-11859] using conventionaltechniques.

EXAMPLE 3 Production of ssAAV Vectors having Codon Optimized Sequences

ssAAV2/8.LSP1.hOTC-co

Plasmids containing the codon optimized hOTCco sequences are cloned asdescribed by replacing the mOTC coding sequencing of the pLSP1mOTCplasmid [Cunningham et al, Mol Ther, 2009, 17: 1340-1346] with thecorresponding cDNA sequence of SEQ ID NO:3, 4, 5, 9 or 10. The resultingplasmids pAAVsc.LSP1.hOTCco are cloned into AAV8 capsids to form thecorresponding ssAAV2/8.LSP1.hOTC-co vectors using techniques describedin Example 1.

B. ssAAV2/rh10.LSP1.hOTC-co

Plasmids containing the codon optimized hOTCco sequences are cloned asdescribed by replacing the mOTC coding sequencing of the pLSP1mOTCplasmid [Cunningham et al, Mol Ther, 2009, 17: 1340-1346] with thecorresponding cDNA sequence of SEQ ID NO:3, 4, 5, 9 or 10. The resultingplasmids pAAVsc.LSP1.hOTCco are cloned into AAV8 capsids to form thecorresponding ssAAV2/8.LSP1.hOTC-co vectors using techniques describedin Example 1.

The vectors generated according to Part A or B may be purified by tworounds of cesium chloride gradient centrifugation, buffered-exchangedwith PBS, and concentrated using Amicon Ultra 15 centrifugal filterdevices-100K (Millipore, Bedford, Mass.). Genome titer (GC/ml) of AAVvectors can be determined by real-time PCR using a primer/probe setcorresponding to the TBG promoter and linearized plasmid standards.Vectors can be subject to additional quality control tests includingsodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)analysis for vector purity and Limulus amebocyte lysate (LAL) forendotoxin detection (Cambrex Bio Science, Walkersville, Md., USA).

EXAMPLE 4

ssAAV8.TBG.hOTCco in Model of Late Onset of OTCD

An AAV8 vector was generated using the methods described herein. Thevector has packaged therein a 5′ AAV2 ITR, a TBG promoter, an intron, ahOTCco, a WPRE element, a bovine growth hormone polyA, and a 3′ AAV2ITR. The expression and kinetics of this vector was compared to aself-complementary AAV8 vector with or without the WPRE element. Theresults show that the single-stranded constructs with the WPRE elementoutperformed those vectors lacking the WPRE element; at comparable dosesboth single-stranded vectors (with and without WPRE) were less robustthan the self-complementary vector lacking WPRE in the time pointsmeasured.

However, the single-stranded vectors may have other desirableproperties, e.g., in terms of kinetics, depending upon the age andcondition of the patient.

EXAMPLE 5 Production of Adenovirus Vectors having Codon OptimizedSequences

hOTCco cDNA [SEQ ID NO: 3, 4, 5, 9 and 10] with NotI linkers is cloneddownstream of a rat PEPCK promoter to generate pPEPCK-hOTC as describedin Mian et al, Molecular Therapy, 2004, 10: 492-499 (2004). This plasmidis digested with Ascl, and the resultant PEPCK-hOTCco fragment isinserted into the adenoviral backbone plasmid pC4HSU31 to generate theparental plasmids pC4HSU-PEPCK-hOTCco. Plasmid pWPRE is digested withClaI to release the WPRE, which is then inserted into the MluI site ofpPEPCK-hOTC, to generate pPEPCK-hOTCco-WPRE plasmid with theirrespective hOTCco sequences. The remaining steps to generate theadenoviral plasmid pC4HSU-PEPCK-hOTCco-WPRE are as previously described.All cloning sites are confirmed by DNA sequence analysis. The identityof recombinant adenoviral plasmids can be confirmed by restrictionenzyme digestion with HindIII and BamHI. The adenoviral plasmids arelinearized with PmeI before transfection into 293Cre4 cells. Adenoviralvectors are rescued and amplified with 293Cre4 cells and helper virusAdLC8cluc. Suspension 293N3Scre8 cells may be used in the final step ofvector production. Purification, quantification by OD260 and viral DNAextraction are performed as described in detail elsewhere[Brunetti-Pierri, N., et al. (2004). Acute toxicity after high-dosesystemic injection of helper-dependent adenoviral vectors into nonhumanprimates. Hum. Gene Ther. 15: 35-46; Ng, P., Parks, R. J. and Graham, F.L. (2002). Preparation of helper-dependent adenoviral vectors. MethodsMol. Med. 69: 371-388].

EXAMPLE 6 Production of hOTCco Lentiviral Vectors

A. Replication-defective lentiviral vectors containing the hOTCcosequences provided herein can be produced by replacing the rat OTC genesequence insert of the plasmid pLenti-GIII-CMV-GFP-2A-Puro [commerciallyavailable from Applied Biological Materials (ABM) Inc.; Canada] with thedesired hOTCco sequence [SEQ ID NO: 3, 4, 5, 9 and 10]. The viruses aregenerated according to manufacturer instructions. The ABM systemincludes an enhancer deletion in the U3 region of 3′ALTR to ensureself-inactivation of the lentiviral vector following transduction andintegration into the target cell's genomic DNA; contains minimallentiviral genes necessary for packaging, replication and transduction(Gag/Pol/Rev), derived from different plasmids all lacking packagingsignals; further, none of the Gag, Pol, or Rev genes are incorporatedinto in the packaged viral genome, thus making the mature virusreplication-incompetent.

B. Replication-defective, non-integrating hOTC Lentiviral Vectors

A DNA construct containing a liver specific promoter and the hOTCco DNAof SEQ ID NO: 3, 4, 5, 9 and 10 are engineered into lentivirus vectorswhich are pseudotyped into sindbis virus E2 enveloped produced asdescribed in US2011/0064763, which is incorporated by reference herein,A11 vectors contain splice donor, packaging signal (psi), aRev-responsive element (RRE), splice donor, splice acceptor, centralpoly-purine tract (cPPT). The WPRE element is eliminated in certainviruses.

C. The hOTCco DNA of SEQ ID NO: 3, 4, 5 9 and 10, is cloned into alentivirus pseudotyped with a vesicular stomatitis virus (VSV) envelopegene, purchased from InvivoGen (SanDiego, Calif.) using manufacturer'sinstructions.

EXAMPLE 7 Production hOTCco RNA Delivery Systems

RNA may be prepared by in vitro transcription from a DNA template orsynthesized. The RNA expression cassette is prepared which includes a 5′UTR, an optional intron with splice donor and acceptor sites, anoptional Kozak sequence, the hOTC coding sequence provided herein, apolyA, and a 3′ UTR using known techniques.

A. A suitable amount of mRNA are incorporated into a lipid-envelopedpH-responsive polymer nanoparticles generated using publishedtechniques. [X. Su et al, Mol. Pharmaceutics, 2011, 8 (3), pp 774-787;web publication: Mar. 21, 2011].

B. Polymeric nanoparticle formulations with 25 kDa branchedpolyethyleneimine (PEI) are prepared as follows. When PEI is present, itmay be branched PEI of a molecular weight ranging from 10 to 40 kDa,e.g., 25 kDa branched PEI (Sigma #408727). Additional exemplary polymerssuitable for the present invention include those described in PCTPublication WO2013182683, the contents of which is hereby incorporatedby reference. The required amount of mRNA is diluted just beforeapplication in water for injection (Braun, Melsungen) to a total volumeof 4 ml and added quickly to 4 ml of an aqueous solution of branched PEI25 kDa using a pipette at an N/P ratio of 10. The solution is mixed bypipetting up and down.

C. For a lipid-based nanoparticle, a lipid formulation is created usingexpression cassette containing the hOTCco RNA in a formulation ofcK-E12:DOPE:Chol:PEG-DMG2K (relative amounts 50:25:20:5 (mg:mg:mg:mg))to provide a solution for delivery. The cationic lipid cK-E12 is used(see, e.g., WO 2013/063468), and is combined withdioleoylphosphatidyl-ethanolamine or “DOPE”, cholesterol (chol), andpolyethylene glycol (PEG) or a PEGylated lipid (PEG-DMG2K) using eformulation methods described in international patent publications WO2010/053572 and WO 2012/170930, both of which are incorporated herein byreference.

EXAMPLE 8 hOTCco DNA Delivery Systems

A. Naked Plasmid DNA—The hOTCco sequences [SEQ ID NO: 3, 4, or 5] areengineered as naked plasmid DNA constructs which are delivered to atarget liver cell (e.g., via intravascular administration) and expressthe human OTC protein in the target cell.

B. Cationic lipid-DNA complexes—Cationic lipid-DNA complexes areprepared using a suitable amount of an expression cassette containing atleast a promoter, an optional intron, an optional Kozak sequences, anhOTCco of SEQ ID NO: 3, 4 or 5, a polyA, and other optional expressioncontrol sequences. The promoter may be a liver specific promoter.Alternatively, another non-tissue specific promoter may be selected. Forexample, a suitable amount of DNA is formulated with a cationic lipid ofcK-E12:DOPE:Chol:PEG-DMG2K (relative amounts 50:25:20:5 (mg:mg:mg:mg))to form a cationic lipid-DNA complex suitable for delivery to a subject.The cationic lipid cK-E12 is used (see, e.g., WO 2013/063468), and iscombined with dioleoylphosphatidyl-ethanolamine or “DOPE”, cholesterol(chol), and polyethylene glycol (PEG) or a PEGylated lipid (PEG-DMG2K)using e formulation methods described in international patentpublications WO 2010/053572 and WO 2012/170930, both of which areincorporated herein by reference.

EXAMPLE 9 Long-term Correction of a Neonatal Lethal Form of OTCDeficiency by Multiple Treatments with AAV Vectors of DifferentSerotypes

In the current study, the scAAV8.TBG.hOTCcoLW4 prepared as described inExample 1 was used to rescue animals in a mouse model of neonatal(early) onset OTCD. OTC KO mice were generated through the deletion ofexons 2-3, and the properties of this mouse characterized in terms ofsimilarity to human patients with null mutations of OTC. In summary, theOTC knockout (KO) model generated in our laboratory through the deletionof exons 2-3 closely mimics the severe neonatal onset form of OTCD inhumans. Neonatal male OTC KO pups have elevated plasma ammonia levelsdue to the absence of OTC expression in the liver, and they inevitablydie within 24 hours after birth. Heterozygous females breed normally,have normal plasma ammonia levels, reduced liver OTC enzyme activity,elevated urine orotic acid levels, and in some cases lower body weightcompared to wild type (WT) littermates. A single injection ofscAAV8-hOTCco vector prepared as described in Example 1 at a dose of1-3×10e10 GC/pup immediately after birth is able to rescue the OTC KOpups and extend the life to 6 weeks. To achieve long-term correction, agroup of 4-week-old OTC-KO mice received a second vector administrationof scAAVrh10-hOTCco vector, which had been prepared as described inExample 1.

Over 30 OTC-KO pups retrieved by Cesarean section have been successfullyrescued with gene delivery. The rescued pups had lower body weight thantheir normal littermates and had a transient phenotype of sparse fur andabnormal skin. Most importantly, their plasma ammonia levels were in thenormal range. However, the efficacy cannot be maintained beyond 6 weeksdue to loss of vector genome during fast liver proliferation in neonatalstage. A second vector administration of scAAVrh10-hOTCco vector in4-week-old OTC-KO mice is able to further extend their lives toadulthood. The oldest mice have reached 18 months of age. The long-termrescued mice show close to normal levels of plasma ammonia, althoughurine orotic acid levels in a subset of these mice were significantlyelevated. Sirius red staining on liver samples from heterozygous mice ofdifferent ages (6, 12, and 18 months old) showed liver fibrosis in aged(18-month old) OTC-KO heterozygous female mice, similar to a liversample from a 11-year-old OTCD patient.

EXAMPLE 10 Treatment of Late Onset OTC Deficiency (OTCD)

Two-month old OTC-KO heterozygous mice received a single tail veininjection of a self-complementary AAV8 vector encoding a codon-optimizedhuman OTC gene (SEQ ID NO: 5) at 1×10¹⁰, 3×10¹⁰ , and 1×10¹¹ vectorgenome copies per mouse. One week following vector treatment, mice inall three vector dose groups had normal urine orotic acid levels whichwere maintained throughout the study (16 months). Liver samples wereharvested from 18 month old treated mice for pathology analysis andcompared to age-matched untreated heterozygous mice and WT littermates.All treated mice showed normal liver histology similar to WT, incontrast to the untreated heterozygous animals which had fibrosisthroughout the liver. In conclusion, a single injection of AAV8sc-hOTCcovector can prevent liver fibrosis in OTC-KO heterozygous and has greatpotential for correction of liver fibrosis in OTCD patients.

Gene therapy vectors described herein are capable of rapid, robust andprolonged gene expression even in mice with a complete lack of OTC.Heterozygous females are able to reproduce and deliver hemizygous maleoffspring, but these pups die within a day of birth if untreated.Untreated old heterozygous female mice show evidence of increasedfibrosis and microvesicular steatosis, a finding that appears similar toobservations in human heterozygous patients. A regimen of gene transferthat is able to rescue affected males has been developed and treatedmales have survived over 72 weeks.

Thus, these data demonstrate that liver-specific gene therapy with hOTCcan prevent liver fibrosis. These data correlate with in treatment ofheterozygous OTC deficient humans, e.g., subjects having late onset ofOTCD.

Sequence Listing Free Text

The following information is provided for sequences containing free textunder numeric identifier <223>.

SEQ ID NO: (containing free text) Free text under <223> 3 EngineeredhOTC 4 Engineered hOTC 5 Engineered hOTC 6 Plasmid pscAAVTBGhOTCLW 8Engineered hOTC 9 Engineered hOTC 10 Engineered hOTC RNA 11 EngineeredhOTC RNA 12 Engineered hOTC RNA 13 Engineered hOTC RNA 14 PCR forwardprimer 15 PCR reverse primer 16 Probe 17 Engineered hOTC cDNA termed LW418 Engineered hOTC cDNA termed LW3

This Application contains a Sequence Listing labeled “14-7037C2USST25.txt” dated Aug. 17, 2020 which is hereby incorporated by reference.The priority U.S. Provisional Patent Application No. 61/950,157, filedMar. 9, 2014, PCT Application No. PCT/US15/19513, filed on Mar. 9, 2015,U.S. patent application Ser. No. 15/122,853, filed on Aug. 31, 2016,U.S. patent application Ser. No. 16/191,709, filed on Nov. 15, 2018, andall published documents cited in this specification are incorporatedherein by reference. Similarly, the SEQ ID NO which are referencedherein and which appear in the appended Sequence Listing areincorporated by reference. While the invention has been described withreference to particular embodiments, it will be appreciated thatmodifications can be made without departing from the spirit of theinvention. Such modifications are intended to fall within the scope ofthe appended claims.

What is claimed is:
 1. A composition comprising a nucleic acid sequenceencoding human ornithine transcarbamylase (hOTCase) and expressioncontrol sequences which direct expression of hOTC in a liver cell,wherein the hOTC nucleic acid sequence is less than 80% identical to thewild-type hOTC sequence over the mature sequence or full length hOTC ofSEQ ID NO:1, and expresses a functional hOTCase, wherein said hOTCnucleic acid sequence is selected from the nucleic acid sequencecomprising SEQ ID NO: 5 or a nucleic acid sequence at least about 96 toabout 99% identical thereto or a nucleic acid sequence selected from SEQID NO: 9, or a nucleic acid sequence at least about 96 to about 99%identical thereto.
 2. The recombinant viral vector according to claim 1,wherein the hOTC nucleic acid sequence has the sequence of SEQ ID NO: 4.3. The recombinant viral vector according to claim 1, wherein the hOTCnucleic acid sequence has the sequence of SEQ ID NO:
 3. 4. Therecombinant viral vector according to claim 1, wherein the hOTC nucleicacid sequence has the sequence of SEQ ID NO:
 8. 5. The recombinant viralvector according to claim 1, wherein the hOTC is a chimeric OTCcomprises a heterologous transit sequence substituted for the nativetransit sequence of SEQ ID NO: 5 or
 9. 6. The recombinant viral vectoraccording to claim 1, wherein the viral vector is selected from anadeno-associated virus (AAV) vector, an adenoviral vector, and alentiviral vector.
 7. The recombinant viral vector according to claim 1,wherein the expression control sequences further comprise aliver-specific promoter.
 8. The recombinant viral vector according toclaim 7, wherein the liver specific promoter is selected from athyroxin-binding globulin (TBG) promoter or a lymphocyte-specificprotein 1 (LSP1) promoter.
 9. The recombinant viral vector according toclaim 1, wherein the expression cassette further comprises one or moreof an intron, a Kozak sequence, a poly A, and a post-transcriptionalregulatory elements.
 10. The recombinant viral vector of claim 1,wherein the recombinant viral vector is a recombinant AAV vector whichcomprises an AA V capsid which has packaged therein a nucleic acidsequence which comprises at least one ITR sequence and the synthetichOTC.
 11. The recombinant viral vector of claim 10, wherein the AAVcapsid is selected from AAV8, AAV9 and/or AAVrh10.
 12. A recombinantadeno-associated virus (rAAV) having an AAV capsid and packaged thereinan expression cassette comprising at least one AAV inverted terminalrepeat (ITR) sequence, an engineered nucleic acid sequence encodinghuman at least the mature ornithine transcarbamylase (hOTCase), andexpression control sequences which direct expression of the hOTCase in aliver cell, said expression control sequences comprising aliver-specific promoter, wherein the hOTC nucleic acid sequence is lessthan 80% identical to the wild-type hOTC sequence over at least themature hOTC of SEQ ID NO: 1 and comprises at least the mature hOTC ofSEQ ID NO: 5 or a nucleic acid sequence at least about 96 to about 99.9%identical thereto or at least the mature hOTC of SEQ ID NO: 9 or anucleic acid sequence at least about 96 to about 99.9% identicalthereto.
 13. The rAAV according to claim 12, wherein the AAV capsid isselected from AAV8, AAV9, or AAVrh10.
 14. The rAAV according to claim12, wherein the expression cassette further comprises a 5′ AAV invertedterminal repeat (ITR) sequence and a 3′ ITR sequence.
 15. The rAAVaccording to claim 12, wherein the at least one AAV ITR comprises a 5′ITR in which the D-sequence and the terminal resolution site is deleted.16. The rAAV according to claim 12, wherein the 5′ and 3′ ITRs are fromAAV2.
 17. The rAAV according to claim 12, wherein the synthetic hOTC hasthe coding sequence of SEQ ID NO:
 3. 18. The rAAV according to claim 12,wherein the synthetic hOTC has the coding sequence of SEQ ID NO:
 4. 19.A viral vector comprising a hOTC gene encoding a chimeric ornithinetranscarbamylase which comprises at least mature human ornithinetranscarbamylase with a heterologous transit sequence, wherein thecoding sequence from the mature human ornithine transcarbamylase isselected from that of a nucleic acid sequence of SEQ ID NO: 3, 4, 5, 8or
 9. 20. A pharmaceutical composition comprising a carrier and aneffective amount of the vector according to claim 1.